TY - JOUR
T1 - Genome editing reveals idiosyncrasy of CNGA2 ion channel-directed antibody immunoreactivity toward oxytocin
AU - Blechman, Janna
AU - Anbalagan, Savani
AU - Matthews, Gary G.
AU - Levkowitz, Gil
N1 - This work was supported by the Israel Science Foundation (No. 1511/16); F.I.R.S.T. (Bikura) Individual Grant (No. 2137/16); Israel Ministry of Agriculture Chief Scientist Office (Nos. 894-0194-13 and 30-04-0002); Minerva-Weizmann program and Adelis Metabolic Research Fund (in the frame of the Weizmann Institute). SA was supported by Israel PBC-VATAT fellowship and by Koshland Foundation. We thank James Trimmer for providing the affinity-purified anti-CNGA5 antibody; Einav Wircer for providing the Tg(oxt:EGFP-oxt3′UTR); Lonnie Wollmuth for useful comments. GL is an incumbent of the Elias Sourasky Professorial Chair.
PY - 2018/9/20
Y1 - 2018/9/20
N2 - Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.
AB - Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.
UR - http://www.scopus.com/inward/citedby.url?scp=85053719506&partnerID=8YFLogxK
U2 - 10.3389/fcell.2018.00117
DO - 10.3389/fcell.2018.00117
M3 - مقالة
SN - 2296-634X
VL - 6
JO - Frontiers in Cell and Developmental Biology
JF - Frontiers in Cell and Developmental Biology
M1 - 117
ER -